The present invention relates to a vector system, such as a lentiviral vector system for the treatment of neurodegenerative disease in a mammal, e.g., Parkinson""s disease.
Further, the present invention relates to a method for treating a neurodegenerative disease and/or symptoms thereof and/or preventing neurodegenerative disease and/or symptoms thereof, in a mammal, comprising, administering a lentiviral vector to a target cell in the brain or nervous system of the mammal, said lentiviral vector comprising a nucleic acid sequence comprising a sequence encoding a growth factor, advantageously in operable linkage with or operably linked to a promoter sequence, wherein said growth factor is expressed in the target cell, thereby treating said neurodegenerative disease. Advantageously the lentiviral vector is primate or non-primate lentiviral vector such as an EIAV vector or an HIV vector or an SIV vector or an FIV vector. Also advantageously, the growth factor is a GDNF, such as a human GDNF. The GDNF can be modified and the nucleic acid molecule encoding the GDNF can likewise be modified; for instance due to the degeneracy of codon usage, the GDNF coding sequence can be modified, and truncated forms of GDNF can be used, such as those which may be found in the literature. Likewise, analogs, homologs, derivatives, and variants of the GDNF coding sequence can be used and ergo of analogs, homologs, derivatives and variants of GDNF can be expressed; advantageously such expressed analogs, homologs, derivatives and variants of GDNF have activity analogous to that of full length human GDNF, e.g., as employed in the exemplified embodiment herein, and the analogs, homologs, derivatives and variants of the GDNF coding sequence encode such active GDNF analogs, homologs, derivatives, and variants. The mammal is advantageously a primate, such as a human. The administration can be by stereotaxic injection. The administration can be intracranially, e.g., intracranially to stiatum or to substantia nigra. The administration can also be by retrograde transport. The neurodegenerative disease can be Parkinson""s disease. The treating of Parkinson""s disease can be by prevention of nigrostriatal degeneration and/or induction of nigrostriatal regeneration and/or reversal of motor deficits. And, the growth factor expression can be for up to 8 months.
Even further still, the lentiviral vector can include additional nucleic sequences, such as nucleic acid sequences encoding one or more other members of the GDNF-family of neurotrophic factors, e.g., neurturin, persphin, neublastin, artemin; and/or the lentiviral vector can contain one or more other nucleotide sequences encoding expression products suitable for treating a neurdegenerative disorder, such as Tyrosine Hydroxylase, GTP-cyclohydrolase I, Aromatic Amino Acid Dopa Decarboxylase and Vesicular Monoamine Transporter 2 (VMAT2), preferably nucleic acid sequences encoding Tyrosine Hydroxylase, GTP-cyclohydrolase I and optionally Aromatic Amino Acid Dopa Decarboxylase or Aromatic Amino Acid Dopa Decarboxylase and Vesicular Monoamine Transporter 2. These other nucleotide sequences may also encode proteins such as growth factors, e.g., NGF (nerve growth factor) and BDNF (brain-derived neurotrophic factor), and antibodies.
Additionally or alternatively, the lentiviral vector encoding the growth factor, e.g., GDNF, can be administered with one or more additional vectors containing one or more additional nucleic acid sequences, such as nucleic acid sequences encoding one or more other members of the GDNF-family of neurotrophic factors, e.g., neurturin, persphin, neublastin, artemin, and/or other nucleotide sequences encoding expression products suitable for treating a neurdegenerative disorder, such as Tyrosine Hydroxylase, GTP-cyclohydrolase I, Aromatic Amino Acid Dopa Decarboxylase and Vesicular Monoamine Transporter 2 (VMAT2), preferably encoding Tyrosine Hydroxylase, GTP-cyclohydrolase I and optionally Aromatic Amino Acid Dopa Decarboxylase or Aromatic Amino Acid Dopa Decarboxylase and Vesicular Monoamine Transporter 2. These other nucleotide sequences may also encode proteins such as growth factors and antibodies. The one or more additional vector can be any suitable vector such as an adenovirus or lentiviral vector; and, it is presently preferred and considered advantageous that the additional vector be a lentiviral vector, as AAV and adenovirus systems, as herein further discussed, do not obtain the enhanced effects observed with the lentiviral-growth factor, e.g., lentiviral-GDNF of the present invention. xe2x80x9cAdministration withxe2x80x9d the lentiviral vector encoding the growth factor, e.g., GDNF, can be through simultaneous administration, e.g., the vectors are admixed in a single formulation that is administered, or via sequential or concomitant administration of the vectors or formulations containing the vectors.
When a vector genome such as a lentiviral or retroviral vector genome comprises two or more nucleic acid sequences (also known as nucleotide sequences of interest or NOIs), it is advantageous that they are operably linked by one or more Internal Ribosome Entry Site(s), e.g., a genome, advantageously a lentiviral genome, comprising three or more NOIs operably linked by two or more Internal Ribosome Entry Site(s) wherein preferably each NOI is useful in the treatment of a neurodegenerative disorder and at least one of the NOIs is a growth factor such as GDNF.
The invention also relates to vector systems, advantageously lentiviral vector systems, used in the methods of the invention, such as a lentiviral vector system which is capable of delivering an RNA genome to a recipient cell, wherein the genome is longer than the wild type genome of the lentivirus, e.g., an EIAV vector system.
According to further aspects of the invention relates to:
a method for producing a lentiviral particle which comprises introducing such a viral genome into a producer cell;
a viral particle produced by such a system or method;
a pharmaceutical composition comprising such a genome, system or particle;
the use of such a genome, system or particle in the manufacture of a pharmaceutical composition to treat and/or prevent a disease;
a cell which has been transduced with such a system;
a method of treating and/or preventing a disease by using such a genome, system, viral particle or cell;
Thus, the invention relates to pharmaceutical compositions comprising the lentiviral vector or the lentiviral vector and other vector(s) employed in the methods of the invention, as well as kits for preparing such compositions (e.g., the lentiviral vector or the lentiviral vector and the other vector(s) in one or more containers and pharmaceutically acceptable excipient, carrier, diluent, adjuvant, and the like in one or more additional containers, wherein said containers can be provided in one or more packages, for instance, packaged together or separately, and optionally including instructions for admixture and/or administration).
The invention can also relate to a bicistronic cassette comprising a nucleotide sequence capable of encoding the growth factor, e.g., GDNF, and an additional nucleic acid sequence, e.g., an additional nucleic acid sequences encoding one or more other GDNF-family of neurotrophic factors or useful in treating or preventing neurodegenerative disease, such as those above-mentioned or otherwise mentioned herein or in documents incorporated by reference herein, operably linked by one or more IRES(s). The invention likewise can also relate to tricistronic cassettes comprising a nucleotide sequence capable of encoding the growth factor, e.g., GDNF, and a first additional nucleic acid sequence, e.g., an additional nucleic acid sequences encoding one or more other GDNF-family of neurotrophic factors or useful in treating or preventing neurodegenerative disease, such as those above-mentioned or otherwise mentioned herein or in documents incorporated by reference herein and a second additional nucleic acid sequence, e.g., an additional nucleic acid sequences encoding one or more other GDNF-family of neurotrophic factors or useful in treating or preventing neurodegenerative disease, such as those above-mentioned or otherwise mentioned herein or in documents incorporated by reference herein, operably linked by two or more IRES(s). Further cassettes are envisioned by the invention.
In addition, it is noted that while herein text may mention employing nucleotide sequences capable of encoding one or more other GDNF-family of neurotrophic factors, e.g., as additional sequences in lentiviral vectors such as lentiviral-growth factor, e.g., lentiviral-GDNF vectors, or in additional vectors administered with lentiviral vectors such as lentiviral-growth factor, e.g., lentiviral-GDNF vectors, such embodiments are not presently preferred because, as discussed below, work involving lentiviral-GDNF-family-gene-neublastin/artemin reported in the literature has not been reproducible.
Further aspects of the invention are described herein.
Parkinson""s disease (PD) is a neurodegenerative disorder characterized by the loss of the nigrostriatal pathway; a progressive disorder resulting from degeneration of dopaminergic neurons within the substantia nigra. Although the cause of Parkinson""s disease is not known, it is associated with the progressive death of dopaminergic (tyrosine hydroxylase (TH) positive) mesencephalic neurons, inducing motor impairment. The characteristic symptoms of Parkinson""s disease appear when up to 70% of TH-positive nigrostriatal neurons have degenerated. Surgical therapies aimed at replacing lost dopaminergic neurons or disrupting aberrant basal ganglia circuitry have recently been tested (C. Honey et al. 1999). However, these clinical trials have focused on patients with advanced disease, and the primary goal of forestalling disease progression in newly diagnosed patients has yet to be realized.
Thus, there is currently no satisfactory cure for Parkinson""s disease or treatments for preventing or treating Parkinson""s disease or its symptoms.
Symptomatic treatment of the disease-associated motor impairments involves oral administration of dihydroxyphenylalanine (L-DOPA). L-DOPA is transported across the blood-brain barrier and converted to dopamine, partly by residual dopaminergic neurons, leading to a substantial improvement of motor function. However, after a few years, the degeneration of dopaminergic neurons progresses, the effects of L-DOPA are reduced and side-effects reappear.
Better therapy for preventing, treating and/or curing Parkinson""s disease and/or symptoms thereof is therefore necessary and desirable.
An alternative strategy for therapy is neural grafting, which is based on the idea that dopamine supplied from cells implanted into the striatum can substitute for lost nigrostriatal cells. Clinical trials have shown that mesencephalic TH positive neurons obtained from human embryo cadavers (aborted foetuses) can survive and function in the brains of patients with Parkinson""s disease. However, functional recovery has only been partial, and the efficacy and reproducibility of the procedure is limited. Also, there are ethical, practical and safety issues associated with using tissue derived from aborted human foetuses. Moreover, the large amounts of tissue required to produce a therapeutic effect is likely to prove to be prohibitive. Some attempts have been made to use TH positive neurons from other species (in order to circumvent some of the ethical and practical problems). However, xenotransplantation requires immunosuppressive treatment and is also controversial due to, for example, the possible risk of cross-species transfer of infectious agents. Another disadvantage is that, in current grafting protocols, no more than 5-20% of the expected numbers of grafted TH positive neurons survive. In order to develop a practicable and effective transplantation protocol, an alternative source of TH positive neurons is required.
A further alternative strategy for therapy is gene therapy: replace dopamine in the affected striatum by introducing the enzymes responsible for L-DOPA or dopamine synthesis (for example, tyrosine hydroxylase); or introduce potential neuroprotective molecules that may either prevent the TH-positive neurons from dying or stimulate regeneration and functional recovery in the damaged nigrostriatal system (Dunnet S. B. and Bjxc3x6rklund A. (1999) Nature 399 A32-A39).
In vivo, dopamine is synthesised from tyrosine by two enzymes, tyrosine hydroxylase (TH) and aromatic amino acid DOPA-decarboxylase (AADC). Parkinson""s disease has been shown to be responsive to treatments that facilitate dopaminergic transmission in caudate-putamen. In experimental animals, genetically modified cells that express tyrosine hydroxylase, and thereby synthesise L-DOPA, induce behavioural recovery in rodent models of PD (Wolff etal. (1989) PNAS (USA) 86:9011-14; Freed et al (1990) Arch. Neurol. 47:505-12; Jiao et al. (1993) Nature 262:4505). However, the functional activity of tyrosine hydroxylase depends on the availability of its cofactor tetrahydrobiopterin (BH4). The level of cofactor may be insufficient in the denervated striatum, and so it is thought that GTP cyclohydrolase I, the enzyme that catalyses the rate limiting step on the pathway of BH4-synthesis, may also need to be transduced to obtain sufficient levels of L-DOPA production in vivo (Bencsics et al (1996) J. Neurosci 16:4449-4456; Leff et al (1998) Exp. Neurol. 151:249-264).
Kaplitt U.S. Pat. No. 6,180,613, and Choi-Lundberg et al. 1997 involve as AAV and Adenoviral vectors, in contrast to the lentiviral vectorsin the present invention, which surprisingly obtain the enhanced effects reported herein such that the work of Kaplitt et al. and Choi-Lundberg et al. fail to teach or suggest the present invention.
C. Rosenblad et al. 2000 reports allegedly using lentiviral vectors to deliver the neublastin/artemin gene. This is a different gene altogether than the gene encoding growth facter GDNF employed in the present invention; and, the neublastin/artemin gene is known not to work and the experiment reported in C. Rosenblad et al. 2000 is not reproducible as the present inventor indeed has tried to do so, and has been unable to reproduce the work of C. Rosenblad et al.
Therefore, although in vivo and ex vivo gene therapy strategies may have been proposed (Dunnet and Bjorklund (1999) as above; Raymon et al (1997) Exp. Neurol. 144:82-91; Kang (1998) Mov. Dis. 13: 59-72) significant progress in this technology has been hampered by many factors, such as the limited efficiency of gene transfer and expression in the target cells, non-reproducible results, and the like. One particular problem in this regard is that the target cells are usually non-dividing cells (i.e. neurones) which are notoriously recalcitrant to transduction. Accordingly, previous therapy strategies fail to teach or suggest the present invention which is herein demonstrated with respect to primatesxe2x80x94mammals close to humans and most indicative of the surprising superiority achieved by the present invention.
WO 98/18934 relates to a polynucleotide sequence for use in gene therapy, which polynucleotide sequence comprises two or more genes operably linked to a promoter, and encodes a fusion protein product of the therapeutic genes. This provides a way of expressing two therapeutic genes from a single xe2x80x9cchimeric genexe2x80x9d. The polynucleotide sequence is capable of encoding a fusion protein comprising tyrosine hydroxylase and DOPA decarboxylase in either TH-DD or DD-TH order, linked by a flexible linker. WO/18924, involves gene transfer systems such as retroviral vectors; and there are other documents that may involve retroviral vectors (See, e.g., Naldini et al., 1996 Science 272, 263; PCT/GB96/01230; Bowtell et al., 1988 J.Virol. 62, 2464; Correll et al., 1994 Blood 84, 1812; Emerman and Temin 1984 Cell 39, 459; Ghattas et al., 1991 Mol.Cell.Biol. 11, 5848; Hantzopoulos et al., 1989 PNAS 86, 3519; Hatzoglou et al., 1991 J.Biol.Chem 266, 8416; Hatzoglou et al., 1988 J.Biol.Chem 263, 17798; Li et al., 1992 Hum.Gen.Ther. 3, 381; McLachlin et al., 1993 Virol. 195, 1; Overell et al., 1988 Mol.Cell Biol. 8, 1803; Scharfman et al., 1991 PNAS 88, 4626; Vile et al., 1994 Gene Ther 1, 307; Xu et al., 1989 Virol. 171, 331; Yee et al., 1987 PNAS 84, 5197; WO99/15683; Verma and Somia (1997) Nature 389:239-242; page 446, Chapter 9 of Coffin et al xe2x80x9cRetrovirusesxe2x80x9d 1997 Cold Spring Harbour Laboratory Press).
WO 98/18934 involves expressing two proteins from a single retroviral vector as a fusion protein (encoded by a single nucleotide sequence) rather than the use of an internal ribosome entry site (IRES) to initiate translation of the second coding sequence in a poly-cistronic message, whereas an IRES, when located between open reading frames in an RNA allows translation of the downstream open reading frame by promoting entry of the ribosome at the IRES element followed by downstream initiation of translation (See WO 9310314).
However, heretofore the use of IRES elements in retroviral vector systems has not been favored because expression of the coding sequence situated downstream of the IRES has often been found to be inefficient, perhaps due to competition for ribosomes and other cellular factors; and, the efficiency of translation initiation would therefore be expected to decrease with increasing numbers of IRES elements.
In addition, another aspect of the art directing against embodiments of the present invention, such as those wherein there is expression of more than one gene, is that there are believed limits on the size of the heterologous gene which can be successfully transduced; and, if incorporation of the heterologous genes and associated regulatory elements dramatically increases the size of the viral genome, then there is a significant risk that it will no longer be able to be successfully packaged, or at least that packaging efficiency will be significantly reduced.
Accordingly, heretofore there has been a need not yet met for gene therapy approaches to treating, preventing, or curing neurodegenerative conditions such as Parkinson""s disease or symptoms thereof; the superior in vivo expression and results obtained therefrom by a lentiviral-growth factor vector, e.g., a lentiviral-GDNF vector, as herein reported, are surprising and unexpected; and, the use of such a vectorxe2x80x94either alone or in combination with other vectors or as a vector that expresses additional productsxe2x80x94especially in treatment, prevention and the like of neurodegenerative conditions such as Parkinson""s disease or symptoms thereof is surprising and unexpected.
Lentiviral delivery of a growth factor, such as glial cell line-derived neurotrophic factor (lenti-GDNF) was tested for its trophic effects upon degenerating nigrostriatal neurons in nonhuman primate models of Parkinson""s disease (PD). Lentiviral-GDNF vectors were injected into the striatum and substantia nigra of nonlesioned aged rhesus monkeys or young adult rhesus monkeys treated 1 week prior with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Extensive GDNF expression with anterograde and retrograde transport was seen in all animals. In aged monkeys, lentiviral-GDNF vectors augmented dopaminergic function. In MPTP-treated monkeys, lentiviral-GDNF vectors reversed functional deficits and completely prevented nigrostriatal degeneration. Additionally, lentiviral-GDNF vectors injections to intact rhesus monkeys revealed long-term gene expression (at least 8 months). In MPTP-treated monkeys, lentiviral-GDNF vector treatment reversed motor deficits in a hand-reach task. These data indicate that GDNF delivery using a lentiviral vector system can prevent nigrostriatal degeneration and induce regeneration in PD and are thus a viable therapeutic strategy for PD patients and patients with neurodegenerative conditions.
Accordingly, the present invention provides a vector system, such as a lentiviral vector system for the treatment of neurodegenerative disease in a mammal, e.g., Parkinson""s disease.
Further, the present invention provides a method for treating a neurodegenerative disease and/or symptoms thereof and/or preventing neurodegenerative disease and/or symptoms thereof, in a mammal, comprising, administering a lentiviral vector to a target cell in the brain or nervous system of the mammal, said lentiviral vector comprising a nucleic acid sequence comprising a sequence encoding a growth factor, advantageously in operable linkage with or operably linked to a promoter sequence, wherein said growth factor is expressed in the target cell, thereby treating said neurodegenerative disease.
Advantageously the lentiviral vector is an EIAV vector or an HIV vector or an SIV vector or an FIV vector.
Also advantageously, the growth factor is a GDNF, such as a human GDNF.
The invention comprehends that the human GDNF can be modified and that the nucleic acid molecule encoding the human GDNF can likewise be modified; for instance due to the degeneracy of codon usage, the human GDNF coding sequence can be modified, and modified and truncated forms of GDNF can be used, such as those which may be found in the literature or analogous to truncated or modified forms found in the literature.
Likewise, analogs, homologs, derivatives, and variants of the human GDNF coding sequence can be used and ergo of analogs, homologs, derivatives and variants of human GDNF can be expressed; advantageously such expressed analogs, homologs, derivatives and variants of human GDNF have activity analogous to that of full length human GDNF, e.g., as employed in the exemplified embodiment herein, and the analogs, homologs, derivatives and variants of the human GDNF coding sequence encode such active GDNF analogs, homologs, derivatives, and variants.
As discussed herein analogs, homologs, derivatives, and variants of human GDNF have homology with GDNF, e.g., at least 75%, preferably at least 85%, more preferably at least 90%, advantageously at least 95%, and more advantageously at least 98% homology with the human GDNF sequence; and, the analog, homolog, derivative and variant advantageously has GDNF activity. One can determine, without undue experimentation, where such variations in human GDNF can be made by comparing, for instance, the GDNFs of various species and noting the differences among them (such differences provide the nature and location of changes to human GDNF that still result in active GDNF), as well as by considering truncated or modified versions of GDNFs that exist in the literature.
Analogs, homologs, variants and derivatives of the sequence encoding human GDNF have at least 75%, preferably at least 85%, more preferably at least 90%, advantageously at least 95%, and more advantageously at least 98% homology; with the human GDNF coding sequence and, the polypeptide advantageously has GDNF activity. One can determine, without undue experimentation, where such variations in the human GDNF coding sequence can be made by considering the degeneracy of codon usage, comparing, for instance, the GDNFs of various species and noting the differences among them (such differences provide the nature and location of changes to human GDNF that still result in active GDNF and the nucleic acid molecule coding sequence can be likewise varied), comparing the nucleic acid molecule coding sequences for GDNFs of various species (as that shows where differences are in coding sequences that still result in a functional GDNF), as well as by considering truncated or modified versions of GDNFs that exist in the literature and nucleic acid molecules encoding such modified or truncated versions of GDNFs.
For instance, an analog, homolog, derivative, or analog of the coding sequence human GDNF can be a nucleic acid molecule that hybridizes specifically to a the coding sequence for human GDNF
As mentioned, an analog, homolog, derivative or variant can be a codon equivalent nucleic acid molecule to the coding sequence for human GDNF. For instance, in generic terms if the invention comprehends xe2x80x9cXxe2x80x9d protein (e.g., human GDNF) having amino acid sequence xe2x80x9cAxe2x80x9d and nucleic acid molecule xe2x80x9cNxe2x80x9d encoding protein X, the invention comprehends nucleic acid molecules that also encode protein X via one or more different codons than in nucleic acid molecule N.
Similarly, the invention envisions polypeptides wherein amino acids are substituted from those of disclosed sequences on the basis of charge and/or structural similarities. That is, in determining suitable analogs, homologs, derivatives or variants of human GDNF, the skilled artisan, without undue experimentation, can consider replacing amino acids in therein with amino acids of similar charge and/or structure so as to obtain a variant, homolog, derivative or variant; and, from making such changes, the skilled artisan can derive a suitable nucleic acid molecule coding sequence for the variant, homolog, derivative, or variant of GDNF, without any undue experimentatioin Thus, the skilled artisan can consider charge and/or structure of human GDNF sequences or portions thereof, in constructing homolgs, variants, analogs and derivatives and nucleic acid molecules coding therefor, without undue experimentation.
In addition, as to nucleic acid molecules encoding human GDNF, the invention comprehends nucleic acid molecules that hybridize under stringent conditions thereto as well as under high stringency conditions thereto and, hybridizing or hybridization under stringent conditions and high stringency conditions can be synonymous with stringent hybridization conditions, terms which are well known in the art; see, for example, Sambrook, xe2x80x9cMolecular Cloning, A Laboratory Manualxe2x80x9d second ed., CSH Press, Cold Spring Harbor, 1989; xe2x80x9cNucleic Acid Hybridisation, A Practical Approachxe2x80x9d, Hames and Higgins eds., IRL Press, Oxford, 1985; both incorporated herein by reference. Thus, specific hybridization of nucleic acid molecules to the nucleic acid molecule encoding human GDNF preferably occurs at stringent hybridization conditions.
One skilled in the art can obtain variants, homologs, analogs or derivatives of human GDNF by PCR, for instance, by PCR amplification of a sample containing a GDNF using a probe or primer or probes or primers that (each) can be any stretch of at least 8, preferably at least 10, more preferably at least 12, 13, 14, or 15, such as at least 20, e.g., at least 23 or 25, for instance at least 27 or 30 contiguous nucleotides in a human GDNF nucleic acid molecule (sequence) which are unique thereto. As to PCR or hybridization primers or probes and optimal lengths therefor, reference is also made to Kajimura et al., GATA 7(4):71-79 (1990).
xe2x80x9cHomologyxe2x80x9d is a well known term. Sequence homology or identity or similarity such as nucleotide sequence homology can be determined using the xe2x80x9cAlignxe2x80x9d program of Myers and Miller, (xe2x80x9cOptimal Alignments in Linear Spacexe2x80x9d, CABIOS 4, 11-17, 1988, incorporated herein by reference) and available at NCBI, as well as the same or other programs available via the Internet at sites thereon such as the NCBI site. Alternatively or additionally, the term xe2x80x9chomologyxe2x80x9d or xe2x80x9cidentityxe2x80x9d, for instance, with respect to a nucleotide or amino acid sequence, can indicate a quantitative measure of homology between two sequences. The percent sequence homology can be calculated as (Nrefxe2x88x92Ndif)*100/Nref, wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence similarity of 75% with the sequence AATCAATC (Nref=8; Ndif=2).
Alternatively or additionally, xe2x80x9chomologyxe2x80x9d or xe2x80x9cidentityxe2x80x9d with respect to sequences can refer to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm (Wilbur and Lipman, 1983 PNAS USA 80:726, incorporated herein by reference), for instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data including alignment can be conveniently performed using commercially available programs (e.g., Intelligenetics(trademark) Suite, Intelligenetics Inc. Calif.). When RNA sequences are said to be similar, or have a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. Thus, RNA sequences are within the scope of the invention and can be derived from DNA sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences.
Additionally or alternatively, sequence identity or similarity or homology such as amino acid sequence similarity or identity or homology can be determined using the BlastP program (Altschul et al., Nucl. Acids Res. 25, 3389-3402, incorporated herein by reference) and available at NCBI, as well as the same or other programs available via the Internet at sites thereon such as the NCBI site. The following references (each incorporated herein by reference) provide algorithms for comparing the relative identity or homology or similarity of sequences such as amino acid residues of two proteins, and additionally or alternatively with respect to the foregoing, the teachings in these references can be used for determining percent homology or identity: Needleman S B and Wunsch CD, xe2x80x9cA general method applicable to the search for similarities in the amino acid sequences of two proteins,xe2x80x9d J. Mol Biol 48:444-453 (1970); Smith T F and Waterman M S, xe2x80x9cComparison of Bio-sequences,xe2x80x9d Advances in Applied Mathematics 2:482-489 (1981); Smith T F, Waterman M S and Sadler J R, xe2x80x9cStatistical characterization of nucleic acid sequence functional domains,xe2x80x9d Nucleic Acids Res., 11:2205-2220 (1983); Feng D F and Dolittle R F, xe2x80x9cProgressive sequence alignment as a prerequisite to correct phylogenetic trees,xe2x80x9d J. of Molec. Evol., 25:351-360 (1987); Higgins D G and Sharp P M, xe2x80x9cFast and sensitive multiple sequence alignment on a microcomputer,xe2x80x9d CABIOS, 5: 151-153 (1989); Thompson J D, Higgins D G and Gibson T J, xe2x80x9cClusterW: improving the sensitivity of progressive multiple sequence alignment through sequence weighing, positions-specific gap penalties and weight matrix choice, Nucleic Acid Res., 22:4673-480 (1994); and, Devereux J, Haeberlie P and Smithies O, xe2x80x9cA comprehensive set of sequence analysis program for the VAX,xe2x80x9d Nucl. Acids Res., 12: 387-395 (1984). And, without undue experimentation, the skilled artisan can consult with many other programs or references for determining percent homology.
Thus, without undue experimentation from this disclosure and the knowledge in the art, the skilled artisan can construct a lentiviral vector containing a coding sequence for a variant, homolog, analog or derivative of human GDNF that encodes and expresses a polypeptide that is functional as a human GDNF, e.g., a variant, homolog, analog, or derivative of human GDNF.
The mammal is advantageously a primate, such as a human.
The administration can be by stereotaxic injection.
The administration can be intracranially, e.g., intracranially to stiatum or to substantia nigra.
The administration can also be by retrograde transport.
The present invention provides the use of a vector system to transduce a target site, wherein the vector system travels to the site by retrograde transport. With respect to retrograde transport, reference is made to UK applications 0122238.9 and 0026943.1 and the corresponding PCT application which claims priority to these UK applications.
The cell body is where a neuron synthesises new cell products. Two types of transport systems carry materials from the cell body to the axon terminals and back. The slower system, which moves materials 1-5 mm per day is called slow axonal transport. It conveys axoplasm in one direction only (from the cell body toward the axon terminals (anterograde transport)). There is also xe2x80x9cFast transportxe2x80x9d which is responsible for the movement of membranous organelles at 50-200 mm per day away from the cell body (anterograde) or back to the cell body (retrograde) (Hirokawa (1997) Curr Opin Neurobiol 7(5):605-614).
Vector systems comprising rabies G protein are capable of retrograde transport (i.e. travelling towards the cell body). The precise mechanism of retrograde transport is unknown, however. It is thought to involve transport of the whole viral particle, possibly in association with an internalised receptor. The fact that vector systems comprising rabies G can be specifically be transported in this manner (as demonstrated herein) suggests that the env protein may be involved.
HSV, adenovirus and hybrid HSV/adeno-associated virus vectors have all been shown to be transported in a retrograde manner in the brain (Horellou and Mallet (1997) Mol Neurobiol 15(2) 241-256; Ridoux et al (1994) Brain Res 648:171-175; Constantini et al (1999) Human Gene Therapy 10:2481-2494). Injection of Adenoviral vector system expressing glial cell line derived neurotrophic factor (GDNF) into rat striatum allows expression in both dopaminergic axon terminals and cell bodies via retrograde transport (Horellou and Mallet (1997) as above; Bilang-Bleuel et al (1997) Proc. Natl. Acd. Sci. USA 94:8818-8823).
Retrograde transport can be detected by a number of mechanisms known in the art. In the present examples, a vector system expressing a heterologous gene is injected into the striatum, and expression of the gene is detected in the substantia nigra. It is clear that retrograde transport along the neurons which extend from the substantia nigra to the basal ganglia is responsible for this phenomenon. It is also known to monitor labelled proteins or viruses and directly monitor their retrograde movement using real time confocal microscopy (Hirokawa (1997) as above).
By retrograde transport, it is possible to get expression in both the axon terminals and the cell bodies of transduced neurons. These two parts of the cell may be located in distinct areas of the nervous system. Thus, a single administration (for example, injection) of the vector system of the present invention may transduce many distal sites.
The present invention thus also provides the use of a vector system where the vector system is or comprises at least part of rabies G to transduce a target site, which comprises the step of administration of the vector system to an administration site which is distant from the target site.
The target site may be any site of interest which is anatomically connected to the administration site. The target site should be capable of receiving vector from the administration site by axonal transport, for example anterograde or (more preferably) retrograde transport. For a given administration site, a number of potential target sites may exist which can be identified using retrograde tracers by methods known in the art (Ridoux et al (1994) as above).
For example, intrastriatal injection of HSV/AAV amplicon vectors causes transgene expression in the substantia nigra, cortex, several thalamic nuclei (posterior, paraventricular, parafasicular, reticular), prerubral field, deep mesencephalic nuclei, mesencephalic grey nucleus, and intrastitial nucleus of the medial as well as dorsal longitudinal fasiculus (Constantini et al (1999) as above).
A target site is considered to be xe2x80x9cdistant from the administrationxe2x80x9d if it is (or is mainly) located in a different region from the administration site. The two sites may be distinguished by their spatial location, morphology and/or function.
In the brain, the basal ganglia consist of several pairs of nuclei, the two members of each pair being located in opposite cerebral hemispheres. The largest nucleus is the corpus striatum which consists of the caudate nucleus and the lentiform nucleus. Each lentiform nucleus is, in turn, subdivided into a lateral part called the putamen and a medial part called the globus pallidus. The substantia nigra and red nuclei of the midbrain and the subthalamic nuclei of the diencephalon are functionally linked to the basal ganglia. Axons from the substantia nigra terminate in the caudate nucleus or the putamen. The subthalamic nuclei connect with the globus pallidus. For conductivity in basal ganglia of the rat see Oorschot (1996) J. Comp. Neurol. 366:580-599.
In a preferred embodiment, the administration site is the striatum of the brain, in particular the caudate putamen. Injection into the putamen can label target sites located in various distant regions of the brain, for example, the globus pallidus, amygdala, subthalamic nucleus or the substantia nigra. Transduction of cells in the pallidus commonly causes retrograde labelling of cells in the thalamus. In a preferred embodiment the (or one of the) target site(s) is the substantia nigra.
In another preferred embodiment the vector system is injected directly into the spinal cord. This administration site accesses distal connections in the brain stem and cortex.
Within a given target site, the vector system may transduce a target cell. The target cell may be a cell found in nervous tissue, such as a neuron, astrocyte, oligodendrocyte, microglia or ependymal cell. In a preferred embodiment, the target cell is a neuron, in particular a TH positive neuron.
The vector system is preferably administered by direct injection. Methods for injection into the brain (in particular the striatum) are well known in the art (Bilang-Bleuel et al (1997) Proc. Acad. Nati. Sci. USA 94:8818-8823; Choi-Lundberg et al (1998) Exp. Neurol.154:261-275; Choi-Lundberg et al (1997) Science 275:838-841; and Mandel et al (1997) ) Proc. Acad. Natl. Sci. USA 94:14083-14088). Stereotaxic injections may be given.
As mentioned above, for transduction in tissues such as the brain, it is necessary to use very small volumes, so the viral preparation is concentrated by ultracentrifugation. The resulting preparation should have at least 108 t.u./ml, preferably from 108 to 1010 t.u./ml, more preferably at least 109 t.u./ml. (The titer is expressed in transducing units per ml (t.u./ml) as titred on a standard D17 cell line). It has been found that improved dispersion of transgene expression can be obtained by increasing the number of injection sites and decreasing the rate of injection (Horellou and Mallet (1997) as above). Usually between 1 and 10 injection sites are used, more commonly between 2 and 6. For a dose comprising 1-5xc3x97109 t.u./ml, the rate of injection is commonly between 0.1 and 10 xcexcl/min, usually about 1 xcexcl/min.
In another preferred embodiment the vector system is administered to a peripheral administration site. The vector may be administered to any part of the body from which it can travel to the target site by retrograde transport. In other words the vector may be administered to any part of the body to which a neuron within the target site projects.
The xe2x80x9cperipheryxe2x80x9d can be considered to be all part of the body other than the CNS (brain and spinal cord). In particular, peripheral sites are those which are distant to the CNS. Sensory neurons may be accessed by administration to any tissue which is innervated by the neuron. In particular this includes the skin, muscles and the sciatic nerve.
In a highly preferred embodiment the vector system is administered intramuscularly. In this way, the system can access a distant target site via the neurons which innervate the innoculated muscle. The vector system may thus be used to access the CNS (in particular the spinal cord), obviating the need for direct injection into this tissue. There is thus provided a non-invasive method for transducing a neuron within the CNS. Muscular administration also enables multiple doses to be administered over a prolonged period.
Another advantage with this system is that it is possible to target particular groups of cells (e.g. sets of neurons), or a particular neural tract by choosing a particular administration site.
In a preferred embodiment, the vector system is used to transduce a neuron which innervates (directly or indirectly) the administration site. The target neuron may, for example, be a motoneuron or a sensory neuron.
Sensory neurons may also be accessed by administration to any tissue which is innervated by the neuron. In particular this includes the skin and the sciatic nerve. Where a patient is suffering from pain (in particular slow, chronic pain), the particular sensory neuron(s) involved in transmitting the pain may be targetted by administration of the vector system directly into the area of pain.
The lentiviral vector is advantageously as in U.S. Pat. Nos. 6,312,683, 6,312,682 or in other patent documents (e.g., applications) incorporated herein by reference wherein the assignee or applicant (e.g., on PCT and UK applications) is Oxford Biomedica, such as UK application serail No 0024550.6 and PCT/GB01/04433, which can also be sources for additional vectors or additional coding sequences to be employed in the practice of the invention, e.g., additional vectors encoding Tyrosine Hydroxylase, GTP-cyclohydrolase I, Aromatic Amino Acid Dopa Decarboxylase and Vesicular Monoamine Transporter 2 (VMAT2) to be employed in the practice of the invention or for coding sequences for Tyrosine Hydroxylase, GTP-cyclohydrolase I, Aromatic Amino Acid Dopa Decarboxylase and Vesicular Monoamine Transporter 2 (VMAT2) to be expressed with the growth factor, e.g., GDNF by the lentiviral vector.
Thus, the present invention comprehends that the administered vector, e.g., the administered lentiviral vector, can include and express additional nucleic sequences, such as nucleic acid sequences encoding one or more other members of the GDNF-family of neurotrophic factors, e.g., neurturin, persphin, neublastin, artemin; and/or the administered vector, e.g., lentiviral vector, can contain and express one or more other nucleotide sequences encoding expression products suitable for treating a neurdegenerative disorder, such as Tyrosine Hydroxylase, GTP-cyclohydrolase I, Aromatic Amino Acid Dopa Decarboxylase and Vesicular Monoamine Transporter 2 (VMAT2), preferably nucleic acid sequences encoding Tyrosine Hydroxylase, GTP-cyclohydrolase I and optionally Aromatic Amino Acid Dopa Decarboxylase or Aromatic Amino Acid Dopa Decarboxylase and Vesicular Monoamine Transporter 2. These other nucleotide sequences may also encode proteins such as growth factors, e.g., NGF and/or BDNF, and antibodies.
Additionally or alternatively, the invention comprehends that the administered vector, e.g., lentiviral vector, encoding the growth factor, e.g., GDNF, can be administered with one or more additional vectors containing one or more additional nucleic acid sequences, such as nucleic acid sequences encoding one or more other members of the GDNF-family of neurotrophic factors, e.g., neurturin, persphin, neublastin, artemin, and/or other nucleotide sequences encoding expression products suitable for treating a neurdegenerative disorder, such as Tyrosine Hydroxylase, GTP-cyclohydrolase I, Aromatic Amino Acid Dopa Decarboxylase and Vesicular Monoamine Transporter 2 (VMAT2), preferably encoding Tyrosine Hydroxylase, GTP-cyclohydrolase I and optionally Aromatic Amino Acid Dopa Decarboxylase or Aromatic Amino Acid Dopa Decarboxylase and Vesicular Monoamine Transporter 2. These other nucleotide sequences may also encode proteins such as growth factors and antibodies. The one or more additional vector can be any suitable vector such as an adenovirus or lentiviral vector; and, it is presently preferred and considered advantageous that the additional vector be a lentiviral vector, as AAV and adenovirus systems, as herein further discussed, do not obtain the enhanced effects observed with the lentiviral-growth factor, e.g., lentiviral-GDNF of the present invention. xe2x80x9cAdministration withxe2x80x9d the lentiviral vector encoding the growth factor, e.g., GDNF, can be through simultaneous administration, e.g., the vectors are admixed in a single formulation that is administered, or via sequential or concomitant administration of the vectors or formulations containing the vectors.
When a vector genome such as a lentiviral or retroviral vector genome comprises two or more nucleic acid sequences (also known as nucleotide sequences of interest or NOIs), it is advantageous that they are operably linked by one or more Internal Ribosome Entry Site(s), e.g., a genome, advantageously a lentiviral genome, comprising three or more NOIs operably linked by two or more Internal Ribosome Entry Site(s) wherein preferably each NOI is useful in the treatment of a neurodegenerative disorder and at least one of the NOIs is a growth factor such as GDNF.
The invention also relates to vector systems, advantageously lentiviral vector systems, used in the methods of the invention, such as a lentiviral vector system which is capable of delivering an RNA genome to a recipient cell, wherein the genome is longer than the wild type genome of the lentivirus, e.g., an EIAV vector system; again, as discussed in documents herein assigned to Oxford Biomedica or wherein Oxford Biomedica is the named applicant, and with the vector expressing a growth factor such as GDNF, e.g., human GDNF, or a variant, homolog, analog or derivative thereof.
According to further aspects of the invention relates to:
a method for producing a lentiviral particle which comprises introducing such a viral genome into a producer cell;
a viral particle produced by such a system or method;
a pharmaceutical composition comprising such a genome, system or particle;
the use of such a genome, system or particle in the manufacture of a pharmaceutical composition to treat and/or prevent a disease;
a cell which has been transduced with such a system;
a method of treating and/or preventing a disease by using such a genome, system, viral particle or cell;
Thus, the invention provides pharmaceutical compositions comprising the lentiviral vector or the lentiviral vector and other vector(s) employed in the methods of the invention, as well as kits for preparing such compositions (e.g., the lentiviral vector or the lentiviral vector and the other vector(s) in one or more containers and pharmaceutically acceptable excipient, carrier, diluent, adjuvant, and the like in one or more additional containers, wherein said containers can be provided in one or more packages, for instance, packaged together or separately, and optionally including instructions for admixture and/or administration).
The invention can also relate to a bicistronic cassette comprising a nucleotide sequence capable of encoding the growth factor, e.g., GDNF such as human GDNF or an analog, homolog, variant or derivative thereof, and an additional nucleic acid sequence, e.g., an additional nucleic acid sequences encoding one or more other GDNF-family of neurotrophic factors or useful in treating or preventing neurodegenerative disease, such as those above-mentioned or otherwise mentioned herein or in documents incorporated by reference herein, operably linked by one or more IRES(s). The invention likewise can also relate to tricistronic cassettes comprising a nucleotide sequence capable of encoding the growth factor, e.g., GDNF such as human GDNF or an analog, homolog, variant or derivative thereof, and a first additional nucleic acid sequence, e.g., an additional nucleic acid sequences encoding one or more other GDNF-family of neurotrophic factors or useful in treating or preventing neurodegenerative disease, such as those above-mentioned or otherwise mentioned herein or in documents incorporated by reference herein and a second additional nucleic acid sequence, e.g., an additional nucleic acid sequences encoding one or more other GDNF-family of neurotrophic factors or useful in treating or preventing neurodegenerative disease, such as those above-mentioned or otherwise mentioned herein or in documents incorporated by reference herein, operably linked by two or more IRES(s). Further cassettes are envisioned by the invention.
In addition, it is again noted that while herein text may mention employing nucleotide sequences capable of encoding one or more other GDNF-family of neurotrophic factors, e.g., as additional sequences in lentiviral vectors such as lentiviral-growth factor, e.g., lentiviral-GDNF vectors, or in additional vectors administered with lentiviral vectors such as lentiviral-growth factor, e.g., lentiviral-GDNF vectors, such embodiments are not presently preferred because, as discussed below, work involving lentiviral-GDNF-family-gene-neublastin/artemin reported in the literature has not been reproducible.
Thus, lentiviral vectors, such as those in Oxford Biomedica patents and patent applications, and vectors for retrograde transport, as in Oxford Biomedica patents and/or patent applications, are advantageous for expression in vivo of a growth factor such as GDNF, e.g., human GDNF or a homolog, variant, analog or derivative thereof; for instance, for treatment, prevention and the like of a neurodegenerative disorder or malady or disease or symptoms thereof, such as Parkinson""s disease.
Accordingly, the neurodegenerative disease can be Parkinson""s disease; and, the invention comprehends methods for treating or preventing Parkinson""s disease or symptoms thereof and compositions therefor and kits for preparing such compositions.
The treating of Parkinson""s disease can be by prevention of nigrostriatal degeneration and/or induction of nigrostriatal regeneration and/or reversal of motor deficits.
And, the growth factor expression, e.g., GDNF such as human GDNF or an analog, homolog, derivative or variant thereof, can be for up to 8 months.
These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.